Enzymes are fundamental components of all living cells. Encoded by genes, and comprising proteins for the most part, enzymes orchestrate the multitude of chemical processes upon which life depends. Simple living organisms, such as bacteria, synthesize around 1000 enzymes, the minimum repertoire for independent existence, while complex organisms such as ourselves synthesize far more, perhaps as many as 30,000. Enzymes are profoundly powerful; the biochemical nuances of a single enzyme among thousands can spell the difference between health or disease; survival or death.
There is considerable interest in enzymes, both as objects for study, and as molecular tools for experimentation and analysis in the laboratory. Chief among the latter are restriction endonucleases, enzymes synthesized by bacteria and archae that catalyze the cleavage of double-stranded DNA molecules. Over two hundred and fifty distinct restriction endonucleases have been discovered, each differing in the specifics of the sites within DNA at which they induce cleavage (Roberts and Macelis, Nucleic Acids Res. 28:306–307 (2000)). Two hundred of these are manufactured commercially, and many have become tools of the trade for molecular biologists in scientific and medical institutions around the world.
Restriction endonucleases bind to duplex DNA at particular sequences of nucleotides termed ‘recognition sequences’. Once bound, they hydrolyze the two sugar-phosphate strands that make up the paired, helical backbones of the DNA molecule. Following hydrolysis, the endonucleases generally detach from the DNA, and the two fragments of the severed DNA molecule are released. As a rule, the two strand-hydrolysis reactions proceed in parallel in a double reaction that requires the presence of two catalytic sites within each enzyme, one for hydrolyzing each strand. In most cases, these two catalytic sites are identical because, in their catalytically active forms at the moment of DNA cleavage, most restriction endonucleases are homodimeric; that is to say, they are composed of two identical protein subunits, or assemblages of subunits, bound to each other in symmetrical, if fleeting, opposition. Each subunit or assemblage possesses a single catalytic site, and so the active homodimer possesses two.
For certain laboratory applications it would be useful if variant restriction endonucleases were available that hydrolyzed only one strand of duplex DNA rather than hydrolyzing both strands. Because such enzymes—‘DNA-nicking’ endonucleases as opposed to ‘DNA-cleaving’ endonucleases—are uncommon in nature, we have developed a method for producing them in the laboratory. This method, and the nicking endonucleases we have made, or anticipate making, by application of the method form the basis for this patent application. The method relies on identifying a restriction endonuclease that possesses two different catalytic sites for strand hydrolysis, rather than two identical sites, and then inactivating one site or the other by mutation to form an altered enzyme in which only one of the two sites retains catalytic ability. Provided the inactivating mutation(s) do not interfere with the functioning of the other catalytic site, or with the ability of the protein to fold properly and bind to its recognition sequence in DNA, the mutated enzyme should hydrolyze only one strand of DNA. Nicking enzymes created in this way will be sequence-specific, in that nicking will occur only at the recognition sequence. They will be strand-specific, in that which of the two DNA strands becomes nicked will depend upon which of the two catalytic sites in the enzyme retains function. And they will be position-specific in that the position of the nick with respect to the recognition sequence, determined by the geometry of the enzyme, will be predictable and constant.